Not applicable.
This invention relates to the field of liposomes. In particular, this invention provides novel liposomes that are pH-sensitive, yet are also stable in serum. The invention further relates to methods of conferring pH sensitivity upon liposomes of varying compositions.
A number of pharmaceutical agents and potential pharmaceutical agents suffer from poor aqueous solubility, high levels of antigenicity, toxicity, or rapid degradation in serum, which can hamper the development of suitable clinical formulations. One solution to these problems has been to encapsulate the pharmaceutical agent in a delivery vehicle that is soluble in aqueous solutions and that shields the agent from direct contact with tissues and blood. In particular, formulations based on liposome technology are of significant interest. Liposomes are vesicles comprised of concentrically ordered phopholipid bilayers which encapsulate an aqueous phase. They form spontaneously by hydrophobic interactions when phospholipids are exposed to aqueous solutions and can accommodate a variety of bioactive molecules.
Liposomes have proved a valuable tool as an in vivo delivery system for enhancing the efficacy of various pharmacologically active molecules (Ostro et al., Liposomes.from Biophysics to Therapeutics, Dekker, New York, pp. 1-369 (1987)). Animal studies have shown that liposomes can decrease the toxicity of several antitumor and antifungal drugs, leading to clinical trials with promising results (Sculier et al., Eur. Cancer Clin. Oncol., 24: 527-538; Gabizon, et al., Eur. J. Cancer Clin. Oncol., 25: 1795-1803 (1989); Treat et al., J. Nati. Cancer Inst., 82: 1706-1710 (1990); Lopez-Berestein et al., J. Infect. Dis., 151: 704-710 (1985); Present et al., Cancer, 62: 905-911 (1988)). In addition, liposomes have been shown to be efficient carriers of antiparasitic drugs for treating intracellular infections of the reticuloendothelial system (RES), in activating macrophage cells to become tumoricidal, in models of metastasis, and in enhancing the immune response to encapsulated antigens, thus facilitating the formulation of artificial vaccines (Liposomes in the Therapy of Infectious Diseases and Cancer, Lopez-Berestein and Fidler, eds. Liss, New York (1989); Alving et al. Immunol. Lett., 25: 275-280 (1990))
Numerous studies have reported on efforts to develop pH-sensitive liposomes as drug delivery systems (Collins, D. in: Liposomes as Tools in Basic Research and Industry (Philippot, J. R. and Schuber, F., Eds.) pp. 201-214, CRC Press, Boca Raton, Fla. (1995) (hereafter, xe2x80x9cCollins 1995xe2x80x9d); see also, Liu, D. et al., Biochim. Biophys. Acta 1022:348-354 (1990); Slepushkin, V. A. et al., J. Biol. Chem. 272:2382-2388 (1997)). Since liposomes are internalized by cells mainly via the endocytic pathway (Straubinger, R. M. et al., Cell 32:1069-1079 (1983)), whereby the liposomes are internalized and exposed to the lowered pH of an endosome, pH-sensitization of liposomes is an attractive strategy to facilitate the delivery of membrane impermeable drugs to the cytoplasm before lysosomal enzymatic degradation occurs. Unsaturated phosphatidylethanolamine (xe2x80x9cPExe2x80x9d) has been widely employed to confer intrinsic pH-sensitivity to liposomes.
At physiological pH in isotonic buffer, the equilibrium phase of unsaturated PE is the inverted hexagonal (HiII) phase (Cullis et al., Biochim. Biophys. Acta 559, 399-420 (1979); Tilcock, C. P. S et al., Biochim. Biophys. Acta 684,212-218 (1982); Allen et al., Biochemistry 29, 2976-2985 (1990)). Under these conditions, PE is protonated and unable to form bilayer (La) vesicles (Papahadjopoulos et al., Biochim. Biophys. Acta 135, 624-638 (1967)). The bilayer phase of unsaturated PE can, however, be stabilized by weakly acidic amphiphiles such as oleic acid (OA) (Straubinger et al., FEBS Lett. 179, 148-154 (1985)) or cholesterylhemisuccinate (CHEMS) (Ellens et al., Biochemistry 23, 1532-1538 (1984)), which confer a negative charge headgroup at pH 7.4. This charge provides electrostatic repulsion to block PE intermolecular interaction/interbilayer contact, thus preventing HII phase formation under physiological conditions. Protonation of the amphipile headgroup caused by a reduction of pH, neutralizes the negative charge and the vesicles become destabilized as the PE component reverts to the HII phase (Litzinger et al., Biochim. Biophys. Acta 11 13, 201-227 (1992). This is generally accompanied by the release of liposomal contents.
Although such liposomes have been shown to be efficient systems for cytoplasmic delivery in cultured cells (Collins 1995, supra), their moderate stability as well as their rapid clearance from the blood have hampered their in vivo use. Despite the fact that small unilamellar PE vesicles (SUV) have been found to be stable in plasma (Liu, et al., Biochemistry 28:7700-7707 (1989); Liu, et al., Biochemistry 29:3637-3643 (1990)), the extraction of the acidic amphipile by the plasmatic albumin results in the rapid loss of the pH-sensitivity. Leventis et al. (Biochemistry 26, 3267 (1987); 3276) and Collins et al. (Biochim. Biophys. Acta 1025, 234-242 (1990) (hereafter xe2x80x9cCollins 1990xe2x80x9d)) demonstrated that the loss of the pH-sensitive moiety can be slowed down by using titratable double-chain amphiphiles such as 1,2-dipalmitoyl-sn-3-succinylglycerol (1,2-DPSG). It was found that although small dioleoylphosphatidylethanolamine (DOPE) liposomes containing 1,2-DPSG maintained their pH-sensitivity after incubation in plasma, there was a substantial shift of the destabilization pH from 5.3 to 4.2 (Collins 1990). Furthermore, after systemic administration, PE liposomes are rapidly cleared from the blood and accumulate in the lung, liver and spleen (Connor et al., Biochim. Biophys. Acta 884:474-481 (1986).
Colloidal stabilization of liposomes can be improved by inclusion of ganglioside (GM1) or poly(ethylene glycol)-derivatized lipids (PEG-PE) (Papahadjopoulos, D. et al., in: Liposomes as Tools in Basic Research and Industry (Philippot, J. R. and Schuber, F., Eds.) pp. 177-188, CRC Press, Boca Raton, Fla. (1995)) (hereafter, xe2x80x9cPapahadjopoulos, 1995xe2x80x9d). These so-called xe2x80x9csterically stabilized liposomesxe2x80x9d (xe2x80x9cSSL,xe2x80x9dor Stealth(copyright) liposomes) have shown long circulation half-lives, reduced uptake by the mononuclear phagocyte system and accumulation in tumors (Papahadjopoulos, 1995;
Woodle, M. C. et al., Biochim. Biophys. Acata 1113:171-199 (1992); Woodle, U.S. Pat. No. 5,356,633). Such coating of PE-based pH-sensitive liposomes increases their stability and circulation time in blood but simultaneously reduces their pH-sensitivity (Liu, D. et al., Biochim. Biophys. Acta 1022:348-354 (1990); Slepushkin, V. A. et al., J. Biol. Chem. 272:2382-2388 (1997)). To circumvent this drawback, the use of cleavable PEG-coating has recently been proposed (Kirpotin, D. et al., FEBS Lett. 388:115‥118 (1997)).
Acid-triggered liposomes destabilization/fusion can be achieved extrinsically by using non-peptidic titratable synthetic polymers (Tirell, D.A. et al., Ann. N. Y. Acad. Sci. 446:237-248 (1985); Kono, K. et al., Biochim. Biophys. Acta -b 1193:1-9(1994)). The advantage of this approach is the potentiality to render different lipid-based formulations sensitive to pH, without the limitations associated with PE-based liposomes. Although fusogenic peptides can also trigger membrane disruption at acidic pH and have been successfully used to enhance the transfection efficiency of plasmid DNA (Wagner, E. et al., Proc. Natl. Acad. Sci. USA 89:7934-7938 (1992)), they display several disadvantages in the development of pH-sensitive liposomes including high cost of production, immunogenicity and non-trivial association to the liposome surface.
Several recent studies have shown that liposomes coated with copolymers of N-isopropylacrylamide (NIPA) bearing alkyl chains, acquire thermo-responsive properties (Wu, X.S. et al., Polymer 33:4659-4662 (1992) (hereafter xe2x80x9cWu, 1992xe2x80x9d); Kono, K. et al., J. Controlled Release 30:69-75 (1994) (hereafter xe2x80x9cKono 1994xe2x80x9d); Kim, J.C. et al., J. Biochem. 121:15-19 (1997) (hereafter xe2x80x9cKim 1997xe2x80x9d)). The alkyl substituent can interact strongly with the liposome membrane and serves as an anchor for the polymers onto the liposomes (Winnik, F. M. et al., Can. J. Chem. 73:2030-2040 (1995); Ringsdorf, H. et al., Biochim. Biophys. Acta 1153:335-344 (1993)). The homopolymer of NIPA is physically characterized by its lower critical solution temperature (xe2x80x9cLCSTxe2x80x9d), which is around 32xc2x0 C. in aqueous solutions (Heskins, M. et al., J. Macromol. Sci. Chem. 2:1441-1455 (1968); Feil, H. et al., Macromolecules 26:2496-2500(1993)). The polymer is soluble below its LCST and separates from solution above it.
This temperature sensitivity was used to destabilize the lipid bilayer of liposomes and to induce the release of their contents in response to an increase in external temperature (Wu 1992; Kono 1994; Kim 1997). By randomly introducing a small amount of a pH-sensitive monomer in the structure of poly(NIPA), it is possible to increase its LCST above 37xc2x0 C. and make the polymer pH-responsive (Taylor, L.D. et al. D. J. Polym. Sci. 13:2551-2570 (1975); Hirotsu, S. et al., J. Chem. Phys. 87:1392-1395 (1987); Chen, G. et al., Nature 373:49-52 (1995)). This property was, for instance, exploited in the preparation of pH-sensitive hydrogels containing cross-linked copolymers of NIPA for the controlled delivery of low molecular weight compounds (Dong, L. C. et al., J. Controlled Release 15:141-152 (1991)) and macromolecular drugs (Kim, Y. H. et al, J. Controlled Release 28:143-152 (1994); Brazel, C.S. et al, J. Controlled Release 29:57-64 (1996)). Hydrogels are, however, constituted of cross-linked polymers, and these finding therefore have no clear application to liposomes, which are formed instead by the hydrophobic interactions of lipid bilayers.
In another study, Wheatley, et al (Proc. Intern. Symp. Control. Rel. Bioact. Mater. 21:600-601 (1994)) changed the amount of poly(ethylacrylic acid) (xe2x80x9cPEAAxe2x80x9d) in buffer containing liposome suspensions and reported that changing the amount of PEAA in the buffer caused corresponding changes in the percentage of leakage of liposomes in the suspension. This approach is not easily translated into in vivo use in a mammal since it would be difficult to maintain and control serum levels of PEAA adequate to act on liposomes (if, indeed, those levels were not themselves harmful to the mammal).
Thus, the art has not succeeded in solving the problem of how to create a pH sensitive liposome which can remain stable in the blood long enough to deliver its contents to target cells.
This invention relates to compositions of liposomes which as pH sensitive, yet serum stable. The invention further concerns the discovery of copolymers that can confer on liposomes, including sterically stabilized liposomes, the ability to release their contents upon a lowering of pH from normal physiological pH to a pH between about 3.5 and about 6.5.
The invention concerns pH sensitive, serum stable liposomes loaded with an agent and having a lipid bilayer, which comprise a thermally-sensitive polymer showing lower critical solution temperature behavior in an aqueous solution, said polymer bearing a hydrophobic substituent and a pH sensitive substituent, wherein said hydrophobic substituent is less than 10 kD and is selected from the group consisting of an alkyl compound and a hydrophobic polymer, which alkyl compound or hydrophobic polymer can insert into the lipid bilayer of the liposome, and further wherein the liposome, when in an aqueous solution, releases at least 20% of the agent upon a change in pH of the solution from pH 7.4 to pH 3.5.
The thermally-sensitive polymer can be NIPA, poly (N-substituted acrylamides, poly(N-acryloyl pyyrolidine), poly(N-acryloyl piperidine, a poly(acryl-L-amino acid amide), a poly(vinyl alcohol) derivative, poly(ethyl oxazoline), hydroxypropyl acrylate, hydroxypropyl cellulose, hydroxyethyl cellulose, polyvinyl acetate phthalate, hydroxypropyl methylcellulose acetate succinate, methylcellulose, hydroxymethyl cellulose, and cellulose.
The alkyl compound can be ODA, a double alkyl chain lipid, or phosphatidylethanolamine. The pH sensitive moiety can be a titratable acidic polymer. In particular, it can be an alkylacrylic acid. Preferred alkylacrylic acids are methylacrylic acid, ethylacrylic acid, propylacrylic acid, and butylacylic acid. The alkylacrylic acid can be present at a mol % of between about 0.5% and about 10%. The liposome can contain PEG. In a preferred embodiment, the thermally-sensitive polymer is NIPA, the alkyl compound is ODA, and the pH sensitive moiety is MAA. Preferred molar ratios of these three molecules are about 94:5:1, about 93:5:2, or about 91:5:4.
The liposome can comprise molecules selected from the group consisting of DSPC, POPC, HSPC, EPC, and EPC/Chol/PEG-PE. In a preferred embodiment, the EPC/Chol/PEG-PE molecules are present in a molar ratio of about 3:2:0.3. The liposomes can be stabilized with PEG, or with ganglioside-derivatized lipids, or with another hydrophilic polymer that stabilizes the liposome and which increases the half life of the liposome in the bloodstream.
The liposome can be loaded with an agent selected from the group consisting of a drug, a radioisotope, a detectable label, a nucleic acid, a vector, and a ribozyme.
The invention further includes methods of delivering an agent to a cell comprising contacting a cell with a liposome of those described above, wherein the liposome is loaded with the agent. The contacting can be caused by systemic administration of the drug. Preferred forms of systemic administration are by injection and by intravenous administration.
The invention further relates to a method of conferring pH sensitivity on, or increasing the pH sensitivity of, a liposome having a lipid bilayer. The method includes the step of complexing the liposome with a thermally-sensitive polymer showing lower critical solution temperature behavior in aqueous solutions, said thermally-sensitive polymer bearing a hydrophobic substituent and a pH sensitive substituent, wherein said hydrophobic substituent is less than 10 kD and is selected from the group consisting of an alkyl compound and a hydrophobic polymer, which alkyl compound or hydrophobic polymer can insert into the lipid bilayer of the liposome, and further wherein the liposome, when in an aqueous solution, releases at least 20% of the agent upon a change in pH of the solution from pH 7.4 to pH 3.5.
The thermally-sensitive polymer can be selected from the group consisting of can be NIPA, poly (N-substituted acrylamides, poly(N-acryloyl pyyrolidine), poly(N-acryloyl piperidine, a poly(acryl-L-amino acid amide), a poly(vinyl alcohol) derivative, poly(ethyl oxazoline), hydroxypropyl acrylate, hydroxypropyl cellulose, hydroxyethyl cellulose, polyvinyl acetate phthalate, hydroxypropyl methylcellulose acetate succinate, methylcellulose, hydroxymethyl cellulose, and cellulose.
The alkyl compound can be ODA, a double alkyl chain lipid, or phosphatidylethanolamine. The pH sensitive moiety can be a titratable acidic polymer. In particular, it can be an alkylacrylic acid. Preferred alkylacrylic acids are methylacrylic acid, ethylacrylic acid, propylacrylic acid, and butylacylic acid. The alkylacrylic acid can be present at a mol % of between about 0.5% and about 10%. The liposome can contain PEG. In a preferred embodiment, the thermally-sensitive polymer is NIPA, the alkyl compound is ODA, and the pH sensitive moiety is MAA. Preferred molar ratios of these three molecules are about 94:5:1, about 93:5:2, or about 91:5:4.
The liposome can contain an agent selected from the group consisting of a drug, a radioisotope, a detectable label, a nucleic acid, a vector, and a ribozyme.
The liposome can comprise molecules selected from the group consisting of DSPC, POPC, HSPC, EPC, and EPC/Chol/PEG-PE. In a preferred embodiment, the EPC/Chol/PEG-PE molecules are present in a molar ratio of about 3:2:0.3.